This invention relates to chambers for carrying out procedures wherein internal atmospheric conditions, such as, e.g., humidity, of the chamber are controlled. More particularly, the invention relates to devices for carrying out steps in the synthesis of a chemical compound and the exiting of fluids such as gases therefrom. The invention has particular application in the manufacture of supports having bound to the surfaces thereof a plurality of chemical compounds, such as biopolymers, which are prepared on the surface in a series of steps.
In the field of diagnostics and therapeutics, it is often useful to attach species to a surface. One important application is in solid phase chemical synthesis wherein initial derivatization of a substrate surface enables synthesis of polymers such as oligonucleotides and peptides on the substrate itself. Support bound oligomer arrays, particularly oligonucleotide arrays, may be used in screening studies for determination of binding affinity. Modification of surfaces for use in chemical synthesis has been described. See, for example, U.S. Pat. No. 5,624,711 (Sundberg), U.S. Pat. No. 5,266,222 (Willis) and U.S. Pat. No. 5,137,765 (Farnsworth).
Determining the nucleotide sequences and expression levels of nucleic acids (DNA and RNA) is critical to understanding the function and control of genes and their relationship to, for example, disease discovery and disease management. Analysis of genetic information plays a crucial role in biological experimentation. This has become especially true with regard to studies directed at understanding the fundamental genetic and environmental factors associated with disease and the effects of potential therapeutic agents on the cell. Such a determination permits the early detection of infectious organisms such as bacteria, viruses, etc.; genetic diseases such as sickle cell anemia; and various cancers. This paradigm shift has lead to an increasing need within the life science industries for more sensitive, more accurate and higher-throughput technologies for performing analysis on genetic material obtained from a variety of biological sources.
Unique or misexpressed nucleotide sequences in a polynucleotide can be detected by hybridization with a nucleotide multimer, e.g., oligonucleotide, probe. Hybridization is based on complementary base pairing. When complementary single stranded nucleic acids are incubated together, the complementary base sequences pair to form double stranded hybrid molecules. These techniques rely upon the inherent ability of nucleic acids to form duplexes via hydrogen bonding according to Watson-Crick base-pairing rules. The ability of single stranded deoxyribonucleic acid (ssDNA) or ribonucleic acid (RNA) to form a hydrogen bonded structure with a complementary nucleic acid sequence has been employed as an analytical tool in molecular biology research. An oligonucleotide probe employed in the detection is selected with a nucleotide sequence complementary, usually exactly complementary, to the nucleotide sequence in the target nucleic acid. Following hybridization of the probe with the target nucleic acid, any oligonucleotide probe/nucleic acid hybrids that have formed are typically separated from unhybridized probe. The amount of oligonucleotide probe in either of the two separated media is then tested to provide a qualitative or quantitative measurement of the amount of target nucleic acid originally present.
Direct detection of labeled target nucleic acid hybridized to surface-bound polynucleotide probes is particularly advantageous if the surface contains a mosaic of different probes that are individually localized to discrete, known areas or sites of the surface. Such ordered arrays containing a large number of oligonucleotide probes have been developed as tools for high throughput analyses of genotype and gene expression. Oligonucleotides synthesized on a solid support recognize uniquely complementary nucleic acids by hybridization, and arrays can be designed to define specific target sequences, analyze gene expression patterns or identify specific allelic variations. The arrays may be used for conducting cell study, for diagnosing disease, identifying gene expression, monitoring drug response, determination of viral load, identifying genetic polymorphisms, analyze gene expression patterns or identify specific allelic variations, and the like.
In one approach, cell matter is lysed, to release its DNA as fragments, which are then separated out by electrophoresis or other means, and then tagged with a fluorescent or other label. The resulting DNA mix is exposed to an array of oligonucleotide probes, whereupon selective binding to matching probe sites takes place. The array is then washed and interrogated to determine the extent of hybridization reactions. In one approach the array is imaged so as to reveal for analysis and interpretation the sites where binding has occurred. Arrays of different chemical probe species provide methods of highly parallel detection, and hence improved speed and efficiency, in assays. Assuming that the different sequence polynucleotides were correctly deposited in accordance with the predetermined configuration, then the observed binding will be indicative of the presence and/or concentration of one or more polynucleotide components of the sample.
The arrays may be microarrays created by in-situ synthesis of biopolymers such as polynucleotides, including oligonucleotides, and polypeptides or by deposition of molecules such as oligonucleotides, cDNA and so forth. In general, arrays are synthesized on a surface of a substrate by one of any number of synthetic techniques that are known in the art. In one approach to the synthesis of microarrays, an apparatus is employed that comprises a reaction chamber and a device for dispensing reagents to the surface of a substrate at discrete sites. A positioning system, which may be a robotic manipulator, moves the substrate to the chamber, in which at least a portion of the device for dispensing reagents is housed. Alternatively, the device for dispensing reagents may be moved in and out of the chamber. A controller controls the application of the reagents to the substrate according to predetermined procedures. The positioning system may comprise one or more stages for moving the substrate to various positions for the dispensing of reagents thereon. The stages may be, for example, an x,y-stepper stage, a theta stage, a rotational stepper stage, and the like.
To produce arrays it is important to reproducibly perform reactions at a particular site without affecting adjacent sites. The reaction should approximate stoichiometry in producing the desired product. Since many of the reactions are performed stepwise, any failure during the synthesis results in the wrong product. The site for each reaction must be defined so that the reaction occurs in a rapid and efficient manner. Each step in the process should provide for a reproducible result and not interfere with the next stage or the reaction at a different site.
Since the arrays provide for a large number of different compounds, the process requires many steps. With oligonucleotides, an in situ synthesis is employed wherein each monomer addition involves a plurality of steps, so that the synthesis at each site involves the number of steps for each addition multiplied by the number of monomers in the oligonucleotide. In order to be able to produce arrays of oligonucleotides efficiently, automated systems are preferred to provide for the accurate placement of reagents, efficient reaction, close packing of different compounds and the indexing of individual oligonucleotides with a particular site in the array.
In situ syntheses generally require a controlled environment in the reaction chamber. For example, many syntheses require an anhydrous environment to avoid the destructive effects of exposing chemical reagents to humidity present in the ambient atmosphere. Typically, an anhydrous chamber is created by enclosing the device for dispensing reagents in a reaction chamber through which dry gas is purged. The gas is delivered into the reaction chamber by means of an inlet, usually a single inlet. Such a technique fails to provide reliably consistent ambient humidity levels because of the turbulent flow that is achieved.
As mentioned above, in certain embodiments of some known reaction chambers, a positioning system, which may be a robotic manipulator, moves the substrate into and out of the reaction chamber through an opening in a wall of the chamber. Generally, the opening serves as an exit for a fluid, usually, a dry gas, that is flowing through the reaction chamber to purge the interior of the chamber in an attempt to provide a moisture-free environment inside the chamber. The opening in the wall is configured in several different ways. In one approach a door is employed. The door may be a pivotally mounted door, a flapper door, a sliding door and the like. In another approach, an air lock transfer station is employed. In operation, the flowing gas exits through the opening in the wall where the door or transfer station provides some degree of protection against air outside the chamber flowing into the interior of the chamber. When it is desired to have the substrate inside the chamber, the door is moved to the open position and the substrate mounted in a suitable holding element is inserted into the interior of the chamber by means of a suitable mechanism such as a robotic arm.
Door-based designs suffer from large-scale flow feature generation near the outlet opening and from highly unsteady flow conditions when the door is opened and closed. For example, if the door is suddenly opened, a region will exist where the boundary layer suddenly separates from the body of the wall surrounding the opening. A separation zone is formed between the exiting gas and the edge, usually, outside edge, of the wall at the opening. Because of very high concentration gradients adjacent the separation zone, outside air can diffuse into the interior of the reaction chamber through this separation zone. The outside air that enters the chamber carries with it moisture from the outside environment, which compromises the anhydrous conditions within the chamber. Furthermore, actuating the door causes fluctuations in pressure. The pressure fluctuations readily initiate three-dimensionality in the flow causing recirculation and subsequent entrainment of moisture into the interior of the chamber.
Other complications resulting from problems with the flow of gas through a chamber include overall low yield in the chemical compounds formed on the surface of the support. Also, undesirable non-uniformity problems with arrays result with arrays because of the very large numbers of features present.
Properly designed air lock transfer stations are able to provide much better protection against the transfer of outside moisture into the interior of the chamber. However, the use of air lock transfer stations significantly lowers throughput speeds. In addition, air lock transfer stations add substantial mechanical and system complexity.
Accordingly, there is a need for a reaction chamber that provides consistent anhydrous conditions within the interior of the chamber. The anhydrous conditions should remain consistent during the insertion and removal of devices into and out of the chamber. Ideally, the reaction chamber should not possess a door or an air transfer station at the opening where the devices are inserted and removed. A steady unidirectional flow of exiting gas through the opening is highly desirable.